sPLA2 inhibitor ester

ABSTRACT

The compound, ((3(2-amino-1,2-dioxoethyl)-2-methyl-1-(phenylmethyl)-1H-indol-4-yl)ox y)acetic acid N-morpholino ester, is disclosed together with its use as a highly bioavailable indole sPLA2 inhibitor compound

FIELD OF THE INVENTION

This application is a 371 of PCT/US 99/08538 filed Apr. 20, 1999.

This invention relates to an sPLA₂ inhibitor compound having highbioavailability.

BACKGROUND OF THE INVENTION

The structure and physical properties of human non-pancreatic secretoryphospholipase A₂ (hereinafter called, “sPLA₂”) has been thoroughlydescribed in two articles, namely, “Cloning and Recombinant Expressionof Phospholipase A₂ Present in Rheumatoid Arthritic Synovial Fluid” bySeilhamer, Jeffrey J.; Pruzanski, Waldemar; Vadas Peter; Plant, Shelley;Miller, Judy A.; Kloss, Jean; and Johnson, Lorin K.; The Journal ofBiological Chemistry, Vol . 264, No. 10, Issue of Apr. 5, 1989; pp.5335-5338, and “Structure and Properties of a Human Non-pancreaticPhospholipase A₂” by Kramer, Ruth M.; Hession, Catherine; Johansen,Berit; Hayes, Gretchen; McGray, Paula; Chow, E. Pingchang; Tizard,Richard; and Pepinsky, R. Blake; The Journal of Biological Chemistry,Vol. 264, No. 10, Issue of Apr. 5, 1989; pp. 5768-5775, the disclosuresof which are incorporated herein by reference.

It is believed that sPLA₂ is a rate limiting enzyme in the arachidonicacid cascade which hydrolyzes membrane phospholipids. Thus, it isimportant to develop compounds which inhibit sPLA₂ mediated release offatty acids (e.g., arachidonic acid) and are highly bioavailable inmammals, especially humans. Such compounds are of value in generaltreatment of conditions induced and/or maintained by overproduction ofsPLA₂; such as septic shock, adult respiratory distress syndrome,pancreatitis, trauma, bronchial asthma, allergic rhinitis, rheumatoidarthritis, etc.

Therapeutic agents that may be given orally are, in general, greatlypreferred and have enhanced commercial potential because of theirinherent ease of use.

U.S. Pat. No. 5,654,326 describes certain indole type sPLA₂ inhibitorsand related ester prodrugs. In particular, this patent exemplifies themethyl ester of((3-(2-amino-1,2-dioxoethyl)-2-methyl-1-(phenylmethyl)-1H-indol-4-yl)oxy)aceticacid.

It is desirable to develop more highly available sPLA₂ inhibitors,particularly those suitable for oral administration.

SUMMARY OF THE INVENTION

This invention is the novel compound,((3-(2-amino-1,2-dioxoethyl)-2-methyl-1-(phenylmethyl)-1H-indol-4-yl)oxy)aceticacid N-morpholino ethyl ester; which is highly bioavailable by oraladministration.

This invention is a pharmaceutical formulation comprising((3-(2-amino-1,2-dioxoethyl)-2-methyl-1-(phenylmethyl)-1H-indol-4-yl)oxy)aceticacid, N-morpholino ethyl ester in combination with a carrier or diluent.

DETAILED DESCRIPTION OF THE INVENTION The 1H-Indole-3-GlyoxylamideCompound of the Invention

The compound of theinvention((3-(2-amino-1,2-dioxoethyl)-2-methyl-1-(phenylmethyl)-1H-indol-4-yl)oxy)aceticacid, N-morpholino ethyl ester; is represented by the structural formula(I);

The N-morpholino ethyl ester (I) is an ester form of known sPLA₂inhibitor((3-(2-amino-1,2-dioxoethyl)-2-methyl-1-(phenylmethyl)-1H-indol-4-yl)oxy)aceticacid, represented by the structural formula (II), below;

The compound of formula (II) is described in Example 1 of U.S. Pat. No.5,654,326 (the disclosure of which is incorporated herein by reference)and European Patent Application No. 95302166.4, Publication No. 0 675110 (publ., Oct. 4, 1995).

It is a discovery of this invention that the compound of formula (I) ishighly bioavailable upon oral administration compared to other sPLA₂inhibitors.

Synthesis of the Compound of the Invention

The synthesis of((3-(2-amino-1,2-dioxoethyl)-2-methyl-1-(phenylmethyl)-1H-indol-4-yl)oxy)aceticacid, N-morpholino ethyl ester (compound of formula I, supra.) uses asstarting material((3-(2-amino-1,2-dioxoethyl)-2-methyl-1-(phenylmethyl)-1H-indol-4-yl)oxy)aceticacid, or a salt thereof (compound of formula II, supra.). This startingmaterial may be prepared by the reaction schemes or method of Example 1of U.S. Pat. No. 5,654,326 (the disclosure of which is incorporatedherein by reference). Similar methods are shown in European PatentApplication No. 95302166.4, Publication No. 0 675 110 (publ., Oct. 4,1995). Other conventional methods may also be used for preparing thestarting material. Procedures useful for the synthesis of the compoundof this invention are specified in Example 1 set out below:

EXAMPLE 1

Preparation of((3-(2-Amino-1,2-dioxoethyl)-2-methyl-1-(phenylmethyl)-1H-indol-4-yl)oxy)aceticacid, N-morpholino ethyl ester, a compound represented by the formula:

Part A

Preparation of N-tert-butoxycarbonyl-3-methoxy-2-methylaniline.

A solution of 44.4 g (344 mmol) of 3-methoxy-2-methylaniline and 75 g(344 mmol)of di-tert-butyl dicarbonate in 400 mL of THF was heated tomaintain reflux for 4 hours. After concentrating at reduced pressure,the residue was taken up in ethyl acetate, washed with iN citric acid,water and dried (MgSO₄). After removing the solvent at reduced pressure,the residue was crystallized from hexane to give 64.5 g (84% yield) ofN-tert-butoxycarbonyl-3-methoxy-2-methylaniline, mp, 56-57° C.

Analysis for C₁₃H₁₉NO₃:

Calculated: C, 65.80; H, 8.07; N, 5.90

Found: C, 63.32; H, 7.83; N, 5.56.

Part B

Preparation of 4-Methoxy-2-methyl-1H-indole.

A solution of 280 mL (0.36 mol) of 1.3M sec-butyl lithium in cyclohexanewas added slowly to N-tert-butoxycarbonyl-3-methoxy-2-methylaniline (43g, 0.18 mol) in 300 mL of THF keeping the temperature below −40° C. witha dry ice-ethanol bath. The bath was removed and the temperature allowedto rise to −20° C. and then the bath replaced. After the temperature hadcooled to −60° C., 18.5 g (0.18 mol) of N-methoxy-N-methylglyoxylamidein an equal volume of THF was added dropwise. The reaction mixture wasstirred 1 hour, the cooling bath removed and stirred an additional 1hour. It was then poured into a mixture of 600 mL of ether and 600 mL of1N HCl. The organic layer was separated, washed with water, dried overMgSO₄, and concentrated at reduced pressure to give 39.5 g of a mixtureof 1-(2-(tert-butoxycarbonylamino)-6-methoxyphenyl)-2-propanone andstarting anilide. This mixture was dissolved in 100 mL of methylenechloride and 40 mL of trifluoroacetic acid and stirred for a total of 26hours. The mixture was washed with water, dried(MgSO₄) and concentratedat reduced pressure. The residue was chromatographed on silica geleluting with 20% EtOAc/hexane to give on crystallization fromCH₂Cl₂/hexane 13.9 g of 4-methoxy-2-methyl-1H-indole, mp, 80-86° C.

Analysis for C₁₀H₁₁NO:

Calculated: C, 74.51; H, 6.88; N, 8.69

Found: C, 74.41; H, 7.08; N, 8.47.

Part C

Preparation of 4-Methoxy-2-methyl-1-(phenylmethyl)-1H-indole.

4-Methoxy-2-methyl-1H-indole (1 g, 6.2 mmol) was added to 248 mg (6.2mmol) of 60% sodium hydride/mineral oil (washed with hexane beforeadding DMF) in 15 mL of DMF and after stirring for 0.5 hour, 0.74 mL(6.2 mmol) of benzyl bromide was added. The mixture was stirred at roomtemperature for 18 hours, diluted with water and extracted with ethylacetate. The ethyl acetate solution was washed with brine, dried (MgSO₄)and after concentrating at reduced pressure, the residue waschromatographed on silica gel eluting with 20% EtOAc/hexane to give 1.3g(84% yield) of 4-methoxy-2-methyl-1-(phenylmethyl)-1H-indole, meltingat 96-116° C.

Analyses for C₁₇H₁₇NO: Calculated: C, 81.24; H, 6.82; N, 5.57 Found: C,81.33; H, 6.74; N, 5.29.

Part D

Preparation of 4-Hydroxy-2-methyl-1-(phenylmethyl)-1H-indole.

A solution of 1.25 g (5 mmol) of4-methoxy-2-methyl-1-(phenylmethyl)-1H-indole and 20 mL of 1MBBr₃/CH₂Cl₂ in 50 mL of methylene chloride was stirred at roomtemperature for 5 hours and concentrated at reduced pressure. Theresidue was dissolved in ethyl acetate, washed with brine and dried(MgSO₄). After concentrating at reduced pressure, the residue waschromatographed on silica gel eluting with 20% EtOAc/hexane to give 577mg (49% yield) of 4-hydroxy-2-methyl-1- (phenylmethyl)-1H-indole,125-127° C.

Analyses for C₁₆H₁₅NO: Calculated: C, 80.98; H, 6.37; N, 5.90 Found: C,80.76; H, 6.26; N, 5.80.

Part E

Preparation of ((2-Methyl-1-(phenylmethyl)-1H-indol-4-yl)oxy)acetic acidmethyl ester.

4-Hydroxy-2-methyl-1-(phenylmethyl)-1H-indole (530 mg, 2.2 mmol) wasadded to 88 mg (2.2 mmol) of 60% NaH/mineral oil in 20 mL of DMF and themixture stirred for 0.67 hours. Then, 0.21 mL (2.2 mmol) of methylbromoacetate was added and stirring maintained for 17 hours. The mixturewas diluted with water and extracted with ethyl acetate. The ethylacetate solution was washed with brine, dried (MgSO₄), and concentratedat reduced pressure. The residue was chromatographed on silica geleluting with 20% EtOAc/hexane to give 597 mg (88% yield) of((2-methyl-1-(phenylmethyl)-1H-indol-4-yl)oxy)acetic acid methyl ester,140-143° C.

Analyses for C₁₉H₁₉NO₃: Calculated: C, 73.77; H, 6.19; N, 4.53 Found: C,74.01; H, 6.23; N, 4.32.

Part F

Preparation of((3-(2-Amino-1,2-dioxoethyl)-2-methyl-1-(phenylmethyl)-1H-indol-4-yl)oxy)acetic acid methyl ester.

Oxalyl chloride (0.16 mL, 1.9 mmol) was added to 582 mg (1.9 mmol) of((2-methyl-1-(phenylmethyl)-1H-indol-4-yl)oxy)acetic acid methyl esterin 10 mL of methylene chloride and the mixture stirred for 1.5 hours.The mixture was concentrated at reduced pressure and residue taken up in10 mL of methylene chloride. Anhydrous ammonia was bubbled in for 0.25hours, the mixture stirred for 1.5 hours and evaporated at reducedpressure. The residue was stirred with 20 mL of ethyl acetate and themixture filtered. The filtrate was concentrated to give 672 mg of amixture of((3-(2-amino-1,2-dioxoethyl)-2-methyl-1-(phenylmethyl)-1H-indol-4-yl)oxy)aceticacid, methyl ester and ammonium chloride, mp 202-215° C.

Part G

Preparation of((3-(2-Amino-1,2-dioxoethyl)-2-methyl-1-(phenylmethyl)-1H-indol-4-yl)oxy)aceticacid.

A mixture of 660 mg (1.7 mmol) of((3-(2-amino-1,2-dioxoethyl)-2-methyl-1-(phenylmethyl)-1H-indol-4-yl)oxy)aceticacid methyl ester and 10 mL of 1N NaOH in 30 mL of methanol was heatedto maintain reflux for 1 hour, cooled to room temperature and stirredfor 0.5 hour. The mixture was concentrated at reduced pressure and theresidue taken up in EtOAc/water. The aqueous layer was separated, madeacidic to pH 2-3 with 1N HCl and extracted with EtOAc. On concentratingthe EtOAc solution, 431 mg (69% yield) of((3-(2-amino-1,2-dioxoethyl)-2-methyl-1-(phenylmethyl)-1H-indol-4-yl)oxy)aceticacid crystallized, melting at 218-220° C.

Analyses for C₂₀H₁₈N₂O₅: Calculated: C, 65.57; H, 4.95; N, 7.65 Found:C, 63.31; H, 4.79; N, 6.91.

Part H

Preparation of((3-(2-amino-1,2-dioxoethyl)-2-methyl-1-(phenylmethyl)-1H-indol-4-yl)oxy)acetic acid N-morpholino ethyl ester.

The compound of the present invention may be formed by the reaction of4-(2-chloroethyl)morpholine hydrochloride (available from AldrichChemical Co., Milwaukee, Wis. USA, Item No. C4,220-3) and suitable basepreferably Cs₂CO₃; and((3-(2-amino-1,2-dioxoethyl)-2-methyl-1-(phenylmethyl)-1H-indol-4-yl)oxy)aceticacid, sodium salt in a suitable solvent, preferably dimethylformamide.The slurry should be heated to 60° C. or other appropriate temperatureuntil a solution is formed. Heating should continued until the reactionis complete. The reaction mixture should be worked up to isolate theproduct using conventional organic laboratory techniques.

Assay 1

Cynomolgus monkeys were used in a single dose pharmacokinetic study. Themonkeys (3 per treatment) were administered a single oral 10 mg/kg doseof one of six indole prodrug compounds including the compound of thisinvention.

Serial blood samples were obtained up to 24 hours after doseadministration. Plasma was analyzed for the corresponding free acidusing an LC/MS method. Also area under the curve (auc) values werecomputed at 8 and 24 hours.

The purpose of this assay was to evaluate and compare the oral deliveryfor selected sPLA₂ inhibitors.

Test Subject:

Species: Monkeys

Strain: Cynomolgus

Dose Preparation:

The amount of sPLA₂ inhibitor was corrected for free acid equivalents.

Vehicle:

Suspension of sPLA₂ inhibitor in 10% Acacia, prepared just prior to doseadministration

Dose Administration:

Route: Oral

Frequency: Single dose

Dose: 10 mg/kg (of the parent acid)

Dosage Volume: 5 mL/kg

TABLE 1 Results Monkey Pharmacokinetics Study AUC AUC Compound Cmax Tmax(0-8 hr) (0-24 hr) ester type (ng/ml) hours ng*h/ml ng*h/ml 1 1604 2.05131 5425 2 200 2.0 1356 2038 3 213 2.0 1277 1761 4 245 2.0 1675 3404 53296 2.0 11919 13161 6 615 3.3 — 8730 1 =((3-(2-amino-1,2-dioxoethyl)-2-methyl-1-(phenylmethyl)-1H-indol-4-yl)oxy)aceticacid N-morpholino ethyl ester 2 =((3-(2-amino-1,2-dioxoethyl)-2-methyl-1-(phenylmethyl)-1H-indol-4-yl)oxy)aceticacid methyl ester 3 =((3-(2-Amino-1,2-dioxoethyl)-1-((1,1′-biphenyl)-2-ylmethyl)-2-methyl-1H-indol-4-yl)oxy)aceticacid N,N-diethylglycolamido ester 4 =((3-(2-Amino-1,2-dioxoethyl)-2-ethyl-1-(phenylmethyl)-1H-indol-4-yl)oxy)aceticacid ethyl ester 5 =((3-(2-Amino-1,2-dioxoethyl)-2-ethyl-1-(phenylmethyl)-1H-indol-4-yl)oxy)aceticacid N-morpholino ethyl ester 6 =((3-(2-Amino-1,2-dioxoethyl)-2-ethyl-1-(phenylmethyl)-1H-indol-4-yl)oxy)aceticacid methyl ester

Assay II

The bioavailability of the compound of the invention,((3-(2-amino-1,2-dioxoethyl)-2-methyl-1-(phenylmethyl)-1H-indol-4-yl)oxy)aceticacid-N-morpholino ethyl ester, was also determined using a Rat Plasmasingle dose oral Pharmacokinetics Study:

The purpose of this assay was to evaluate and compare the oral deliveryfor selected sPLA₂ inhibitors.

Test Subject:

Species: Rat

Strain: Fischer 344

Dose Preparation:

The amount of sPLA₂ inhibitor was corrected for free acid equivalents.

Vehicle:

Suspension of sPLA₂ inhibitor in 10% Acacia, prepared just prior to doseadministration

Dose Administration:

Route: Oral

Frequency: Single dose

Dose: 10 mg/kg (of the parent acid)

Dosage Volume: 5 mL/kg

Rats fasted overnight.

Specimen Collection:

Blood samples (0.8 ml) were obtained at the following times: 0.5, 1, 2,4, 6 and 8 hours (2 rats/timepoint)

Data Analysis:

Plasma was assayed by HPLC to measure concentrations of the differentsPLA₂ inhibitors (as free acids).

Cmax (maximal plasma concentrations), and AUC values were determinedfrom the mean plasma concentration-time profiles.

TABLE 2 Compound Cmax AUC ester type (ng/ml) (0-8 hr) 11 1094 2400 12 79385 13 258 1229 14 1199 2604 15 612 1504 16 259 1031 11 =((3-(2-amino-1,2-dioxoethyl)-2-methyl-1-(phenylmethyl)-1H-indol-4-yl)oxy)aceticacid N-morpholino ethyl ester (compound of the invention) 12 =((3-(2-Amino-1,2-dioxoethyl)-2-methyl-1-(phenylmethyl)-1H-indol-4-yl)oxy)aceticacid N,N-diethylacetamido ester 13 =((3-(2-Amino-1,2-dioxoethyl)-2-methyl-1-((1-naphthalenyl)methyl)-1H-indol-4-yl)oxy)aceticacid N,N-diethylacetamido ester 14 =((3-(2-Amino-1,2-dioxoethyl)-2-methyl-1-((1-naphthalenyl)methyl)-1H-indol-4-yl)oxy)aceticacid morpholino N-ethyl ester 15 =((3-(2-Amino-1,2-dioxoethyl)-2-ethyl-1-((3-chlorophenyl)methyl)-1H-indol-4-yl)oxy)aceticacid N-morpholino ethyl ester 16 =((3-(2-Amino-1,2-dioxoethyl)-2-ethyl-1-((3-chlorophenyl)methyl)-1H-indol-4-yl)oxy)aceticacid N,N-diethylacetamido ester

While the present invention has been illustrated above by certainspecific embodiments, it is not intended that these specific examplesshould limit the scope of the invention as described in the appendedclaims.

We claim:
 1. The compound,((3-(2-amino-1,2-dioxoethyl)-2-methyl-1-(phenylmethyl)-1H-indol-4-yl)oxy)aceticacid N-morpholino ethyl ester.
 2. A pharmaceutical formulationcomprising the compound of claim 1 in combination with a carrier ordiluent.